Recent advances in genetic engineering have enabled the development of new biological platforms for the production of molecules, heretofore only synthesized by chemical routes. Although microbial fermentation is routinely exploited to produce of small molecules and proteins of industrial and/or pharmaceutical importance (antibiotics, enzymes, vaccines, etc.), the possibility of using green plants to manufacture a high volume of chemicals and materials has become an increasingly attractive alternative.
Using green plants to produce large amounts of compounds has two significant advantages over traditional chemical synthesis. First, green plants constitute a renewable energy source, as opposed to finite petrochemical resources. Because of photosynthesis, the only raw materials that are required to produce carbon-based compounds in green plants are carbon dioxide, water, and soil. Sunlight is the ultimate source of energy. Second, in comparison to existing fermentation facilities that are expensive and limited in size, green plants constitute a huge available biomass that could easily accommodate the large amounts of chemicals that are required for certain high-volume, low-cost applications.
Producing para-hydroxybenzoic acid in green plants transformed with 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) has been previously described (Mayer et al., Plant Cell, 13:1669-1682 (2001) and U.S. Ser. No. 10/359,369). Mitra et al. (PLANTA, 215:79-89 (2002)) express an HCHL in hairy root cultures of Datura stramonium. Expression of HCHL enzymes in plant cells leads to production of para-hydroxybenzoic acid (pHBA) from 4-coumaroyl-CoA (pHCACoA). The pHBA produced in plants is rapidly glucosylated by one or more endogenous UDP-glucosyltransferases into pHBA glucosides (both phenolic and ester glucosides) (Mayer et al., supra; Mitra et al., supra, and U.S. Ser. No. 10/359,369) that are subsequently sequestered in the plants' vacuoles.
pHCACoA is normally used by plants to make molecules that are secondary metabolites with roles as plant growth regulators, UV protectants, or cell wall components such as lignin, cutin, or suberin. Examples of secondary metabolites made from pHCACoA include caffeoyl-CoA and feruloyl-CoA. Expression of HCHL genes in tobacco plants under the control of a constitutive promoter (CaMV35S) leads to plant growth defects such as interveinal leaf chlorosis, stunting, low pollen production, and male sterility (Mayer et al., supra). As a result of constitutive HCHL expression (in all plant tissues), pHCACoA levels were depleted to a point where molecules derived from pHCACoA that are essential for plant growth and reproduction were no longer produced in adequate amounts.
HCHL expression needs to be targeted to cells where suitable pools of pHCACoA exist and where conversion to pHBA does not detrimentally affect plant growth and reproduction. Plant stem tissue contains a significant pool of available pHCACoA and can accommodate large fluxes to the phenylpropanoid pathway. In order to exploit the available substrate pool without causing detrimental effects to the plant, HCHL expression needs to be limited to plant stem tissue. In addition, expression levels need to be high enough to produce suitable quantities of pHBA. Robust tissue-specific plant promoters, namely those which are known to drive genes involved in cell wall biosynthesis, represent an attractive group of candidate promoters for HCHL expression.
Genes involved in the production of phenylpropanoid derivatives used in plant cell wall biosynthesis (which are expected to show a tissue-specific expression pattern) represent a source of possible promoters to drive tissue-specific HCHL expression. Examples of these genes include cinnamate-4-hydroxylase (C4H; GenBank® U71080), 4-coumaroyl-Coenzyme A ligase (4CL1; GenBank® U18675), para-coumarate 3-hydroxylase (C3′H; AC011765), and the genes encoding proteins responsible for the catalytic activity of cellulose synthase (IRX1, IRX3, IRX5, and their respective orthologs from rice and maize)(Taylor et al., PNAS, 100(3):1450-1455 (2003)). Given the requirement that HCHL expression must be limited to stem tissue, it is unknown if any of these promoters are suitable for stem-specific expression. Use of these promoters for HCHL expression in plant stalk tissue has not been reported.
Cellulose is a polymer of β(1,4)-linked glucose. It is an essential component of both the primary and secondary cell walls in higher plants.
Cellulose can make up to 90% of the dry weight of the secondary walls. In the plant cell wall, individual cellulose chains crystallize to form microfibrils. Cells involved in synthesizing the cellulose for the secondary cell wall represent an attractive target for tissue-specific expression of HCHL.
Cellulose synthesis is believed to involve a multienzyme complex situated at the plasma membrane (Taylor et al., Plant Cell, 11 (5):769-779 (1999); Taylor et al., supra (2003)). Many of the cellulose synthase genes “CesA genes” are classified as such based on highly-conserved motifs (Richmond and Sommerville, Plant Physiol., 124:495-498 (2000) and Delmer, D P, Annu. Rev. Plant Physiol. Plant Mol. Biol., 50:245-276 (1999)). Many of the genes share homology with one another, yet appear to have different roles in cellulose biosynthesis. The CesA genes are a subset of a larger family of related genes which share some homology to one another. These genes form a family of cellulose synthase-like genes (“csl” genes; Taylor et al., supra (2003); Richmond, T., Genome Biol., 1 (4):reviews 3001.1-3001.6 (2000)) whose exact function is not known.
Use of promoters from CesA genes have previously been described. Turner et al. (WO 00/070058) describe the use of cellulose synthase genes or promoters (IRX3) for modulating enzymes involved in the synthesis of plant cell walls. Jones et al. (Plant Journal, 26(2):205-216 (2001)) described the utility of the IRX3 promoter to down-regulate genes involved with lignin synthesis in plant stalk tissue. Allen et al., (WO 00/04166) describe methods related to altering cellulose synthase genes (CesA). Stalker et al. (WO 98/18949) describe a CesA homolog from cotton (Gossypium hirsutem) and methods associated with altering cotton fiber and wood quality. Arioli et al. (WO 98/00549) describe methods for manipulating a cellulose synthase-like gene (rsw1) for altering cellulose biosynthetic properties. None of these references teach the use of a cellulose synthase-like gene promoter to drive HCHL expression.
The IRX3 gene was putatively identified as encoding the cellulose synthase catalytic subunit from Arabidopsis (Turner et al., Plant Cell, 9(5): 689-701 (1997). Expression of the IRX3 gene was shown to be normally limited to plant stem tissue as no detectable mRNA transcript was measured in leaf tissue (Taylor et al., supra (1999)). It was later reported that the catalytic activity of cellulose biosynthesis is attributed to a multi-subunit complex formed by the proteins encoded by the IRX1, IRX3, and IRX5 genes (Taylor et al., Plant Cell, 12:2529-2539 (2000) and Taylor et al., supra (2003)). These three genes identified from Arabidopsis show essentially the same expression patterns. Expression of these genes is normally limited to cells involved in secondary cell wall biosynthesis. Additionally, orthologs of these genes may exhibit similar tissue-specific expression patterns, namely expression in cells that produce cellulose for secondary cell wall synthesis. The prior art does not teach use of the promoters from IRX1, IRX3, or IRX5 (or orthologs thereof) for stem tissue expression of HCHL.
The problem to be solved is to identify regulatory sequences that allow targeted HCHL expression in plant tissues where significant pHBA accumulation can occur without adversely affecting the synthesis of compounds essential for plant growth and development. In other words, technology needs to be developed that allows for HCHL-mediated pHBA production in plants without negative effects on plant performance in the field.